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infiltrated patch overlapped with the first one. The two infiltrations were carried
out at a 5-day interval. The rationale was that if a localized GFP silencing signal
had moved from the first infiltrated patch to adjacent, non-transgenic cells, GFP
expression from the second overlapping patch would be prevented at the edge of
the first one. In the absence of cell-to-cell signalling from the first patch, however,
expression of GFP from the second patch would coincide with the edge of the first
patch (see Plate 3.5, following page 146). The outcome of those experiments showed
that localized signalling indeed occurred in non-transgenic leaves and that its extent
was the same as in leaves of the GFP transgenic N. benthamiana . Moreover, as in
the GFP transgenic N. benthamiana , this process was abolished if the first patch
had received a P19 co-treatment (Himber et al. , 2003). Therefore, unlike systemic
movement of Nia silencing, the cell-to-cell movement of GFP silencing did not
require prior transcription of a homologous transgene in recipient tissues.
Molecular analysis of the cell-to-cell movement of GFP silencing outside of the
Arabidopsis vasculature was also carried out in wt, sde1 and sde3 mutant back-
grounds (Himber et al. , 2003). In these plants, the pan-handled transgene construct
used to trigger GFP silencing in the phloem companion cells corresponded to the
5 part of the GFP cDNA ('GF'). In all three genetic backgrounds there were com-
parable levels of 21 nt and 24 nt 'GF' siRNAs, despite very dissimilar silencing
movement phenotypes. Thus, accumulation of the primary 'GF' siRNAs produced
into the phloem was not correlated with the extent of silencing movement in the
lamina. 'P' siRNAs originating from the non-overlapping part between the silenc-
ing trigger and the GFP transgene were then analysed. These secondary siRNAs are
diagnostic of transitivity. It was found that in contrast to the 'GF' siRNAs, accumu-
lation of the 'P' siRNAs was strictly correlated to the extent of silencing movement.
Hence, high levels of 'P' siRNAs in wt plants were linked to full silencing, lower
levels of 'P' siRNAs in the sde3 mutant correlated with intermediate movement of
silencing whereas the complete lack of 'P' siRNAs in the sde1 mutant was associated
with the vein-centred movement of silencing over 10-15 cells only. Interestingly, the
secondary 'P' siRNAs were found to be exclusively of the 21 nt size class, indicating
that the dsRNA product of transitivity is processed by a specific Dicer that generates
21 nt siRNAs only (Himber et al. , 2003). Thus, extensive movement is linked to the
recruitment, by SDE1 and SDE3, of GFP mRNA in recipient cells for the production
of 21 nt long secondary siRNAs. By contrast, short-distance silencing movement is
independent of SDE1 and SDE3, does not require the presence of GFP mRNA in
recipient cells and can occur in the absence of the 24 nt siRNA at the site of initiation.
3.3.5
Maintenance of systemic silencing
Grafting involving the Nia transgenic plants showed that tissues of Class-I, Class-II
and Class-III plants were all competent to perceive the systemic silencing signal and
activate the sequence-specific degradation of Nia transcripts. However, de-grafting
experiments indicated that Class-I and Class-III plants were unable to sustain pro-
duction of the systemic signal, as they progressively lost the silenced phenotype in the
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